Measurement of the conditions or the presence of substances in an environment by detection of the rate of hydrolysis of an agent is well known. Specifically, the use of fluorescent substrates for such measurements is known, although such use is typically not practicable for measurements of very low levels of hydrolyzing agents, such as enzymes. Generally, by removal of a chemical moiety, the fluorescence of the compound increases. The prior compounds have been unsuitable, however, for measurement of low levels of enzyme in an aqueous environment.
All references herein cited are herein incorporated by reference.
A. Enzyme Measurement
Generally, the measurement of alkaline phosphatase (AP) has been used in diagnosis of various diseases because of its ubiquitous presence in the cell membranes of tissues in the body. Fernley, N. H., Mammilian Alkaline Phosphatases, in: Boyer, P. D. (ed.), The Enzymes, Vol. IV, Academic Press, New York 1971, pp. 417-447. Various esterases are also measured for clinical diagnoses of disease. Bergmeyer, H. U., Methods of Enzymatic Analysis, 3d.ed., Vol. IV, 1-143, Verlag Chemie, 1984. With recent advances in biological techniques, these enzymes could be used as markers in combination with biological probes for the detection of complementary biological molecules. Thus, the activity of the enzyme indirectly measures the quantity of the biological substance complementary to the probe. While various esterases may be used, alkaline phosphatase measurement provides a convenient assay for the detection and measurement of complementary biological molecules. Bergmeyer, supra, at Vols, 10-11.
Previously, the level of AP has been monitored using UV visible spectrophotometry, radioimmunoassay (RIA), and fluorescent substrates.
UV compounds have been tried for assays of AP, for example thymolphthalein monophosphate, Coleman, C. M., Clin. Chim. Acta, 13:401 (1966), phenolphthalein monophosphate, Wilkerson, J. H. and Vodden, A. V., Clin. Chem., 12:701 (1966) and para-nitro-phenylphosphate, Neuman, H. and Van Vrudendaal, M., Clin. Chim. Acta, 17:183 (1967). These compounds are approximately a thousand times less sensitive than efficient fluorescent compounds necessary to determine 10 attomole/mL (10.times.10.sup.-18 moles/mL) of AP.
There has been a variety of fluorescent substrates described in literature which have been used in assaying AP. None of these substrates have been entirely satisfactory for a variety of reasons.
7-Hydroxycoumarin phosphate, Glazer, R. and Haynes, M., Anal. Letters, 1 (5):333-45 (1968) and Sherman, William R. and Robine, Eli, Anal. Chem, 40/4:803-51 (1968), requires a high substrate level of 10 mM to saturate the enzyme. Its Stokes' shift is 78 nm (excitation of 376 nm and emission of 454 nm) and the Raman fluorescence of 422 nm. Since the Raman is close to the fluorescence maximum it can mask the signal being generated at 454 nm. Thus, these factors would have adverse effects on the ability of the substrate to measure very low levels of AP rapidly.
2-Benzoxazolyl-7-hydroxycoumarin phosphate, Wolfbeis, Otto S. and Koller, Ernst, Mikrochemica Acta, 389-95 (1985) has a low Stokes' shift of 44 nm (excitation of 427 nm with an emission of 471 nm). A second negative associated with this substrate is its low aqueous solution stability in tris pH 9.5 even at -15.degree. C.
2-Phenyl-7-hydroxycoumarin phosphate, Otto S. and Koller, Ernst, Mikrochemica Acta, 389-95 (1985) has a higher Stokes' shift of 88 nm (excitation of 383 nm with an emission of 471 nm), however it has poor aqueous solution stability, as noted for the previous compound.
Fluorescein phosphate has a Stokes' shift of 25 nm (excitation of 490 nm and emission of 515 nm). This small Stokes' shift makes it completely unsuitable for the determination of AP at low levels. See, Tiffany, T. O.; Watsky, M. B.; Burtis, C.a. and Thacker, L. H., Clin. Chem., 19/8:871-82 (1973).
3-Hydroxy-2-naphthanilide-6-bromo, 3-hydroxy-2-naphthyl-o-anisidine phosphate, Guilbault, G. G., Newer Fluorometric Methods for the Analysis of Biologically Important Compounds, In: Fluorescence Techniques in Cell Biology, Thaer, A. A. and Sernetz, M., ed., Springer-Verlag, N. Y., Heidelberg, Berlin, 235-42 (1983); Vaughn, A.; Guilbault, G. and Hackney, D., Anal. Chem., 43/6:721-4 (1971) and Guilbault, G. G., J. Res. NBA, 76A/6:607-12 (1972), has a Stokes' shift of 110 nm (excitation of 405 nm and emission of 515 nm). However, the 1971 reference noted that there is a residual fluorescence at 515 nm due to the remaining phosphorylated substrate which would reduce the ultimate sensitivity of the substrate. Also, this substrate has only been used in a solid surface assay in intact cells for microscopic visualization of the presence of AP. Because of the structure of this substrate it is likely that the hydrolyzed product would be insoluble under the basic aqueous conditions of assays of biological material. This would limit the usefulness of the substrate.
2-Hydroxy-3-naphthoic anilide phosphate, Tsou, K. C. and Matsukawa, Sadao, J. Med. Chem., 11/15:1097-9 (1968) has a Stokes' shift of 220 nm (excitation of 300 nm and emission of 520 nm). However this substrate has only been used in a histochemical assay system. The hydrolyzed product, 2-hydroxy-3-naphthoic acid anilide, has a low solubility which could complicate its use in a kinetic or end point assay. Also, its background fluorescence is reported to be relatively high, at 520 nm indicating its ultimate sensitivity might be low due to the high background reading.
3-0-Methylfluorescein phosphate (3-0-MFP), Hill, Hoyle D., Summer, George K., and Waters, Michael D., Anal. Biochem., 24:9-17 (1968); Wolfbeis, Otto S. and Koller, Ernst, Mikrochemica Acta, 389-95 (1985); Hashimoto, Shinya, Kobayashi, Kensei; Fujiwara, Kitao, Harabuchi, Hiroki and Fuwa, Keiichiro, Bunseki Kagaku, 32:E177-E184 (183) and Norgaard, Aage, Kjeldsen, Keld, Larsen, Jim Stenfatt; Larsen, Christian Gronhoj and Larsen, Frederik Gronhoj, Scand. Clin. Lab. Invest., 45/2:139-44 (1985) has a Stokes' shift of 15 nm. Hashimoto, et al., have also noted problems of hydrolysis of the phosphate under aqueous conditions suitable for the assay of AP.
Riboflavin-5-phosphate, Glazer, R. and Haynes, M., Anal. Letters, 1(5):333-45 (1968) and Takeuchi, T. and Nogami, S., Acta Pathol. Japan, 4, 277 (1954) has been used in tissue AP assays, only. The ultimate sensitivity of the assay has not been reported.
Flavone disphosphate has an emission wavelength of 510 nm. Glazer, R. and Haynes, M. Anal. Letters, 1 (5):33-45 (1968) and Land, D. B. and Jackim, E. Anal. Biochem., 16:481 (1966). The excitation wavelength was not reported. The authors reported that it was a more stable substrate than 3-0-MFP and a more sensitive fluorescence indicator than beta-naphthol phosphate. This substance requires the removal of two phosphate groups before the initiation of fluorescence can be observed. This would cause severe problems for a kinetic assay in which only a fraction of the starting substrate is converted to monophosphate which is not fluorescent. Then the monophosphate would have to be converted to the 3-hydroxy-flavone before the fluorescence emission could be observed.
4-methyl umbelliferyl phosphate (4-MUP), Wolfbeis, Otto S. and Koller, Ernst, Microchemical Acta, 389-95 (1985); Cornish, Coralie J., Neale, Francis C. and Posen, Solomon, Amer. J. Clin. Pathol., 53/1:68-76 (1970) and Sherman, William R. and Robine, Eli, Anal. Chem., 40/4:803-5 (1978), has a Stokes' shift of 82 nm with an excitation at 367 nm. Emission is 449 nm. The first order Raman is 416 nm which is 1/120 that of 4-methyl-umbelliferone (4-MU). The emission contributes to a high background fluorescence. Cornish et al. report that 4-MUP has an emission at 465 nm, which is 1/120 that of 4-methyl-umbelliferone (4-MU). It was also noted by these authors that the 4-MUP breaks down in basic tris buffer. They were able to decrease this hydrolysis problem by preparing the 4-MUP in a bicarbonate/carbonate buffer. Hashimoto, Shinya, Kobayashi, Kensei, Fujiwara, Kitao, Harabuchi, Hiroki and Fuwa, Keiichiro, Bunseki Kagaku, 32:E177-E184 (1983) reported that their survey of the literature showed that 4-MUP "seems the most promising substrate for further investigation on the dissolved enzymes (AP) in natural waters. Using this substrate with 48 hours incubation, the lowest limit of the determination of AP activity was 1.times.10.sup.-12 moles 1.sup.-1 min.sup.-1. On the other hand, that of conventional spectrophotometric method using p-NPP was 0.4.times.10.sup.-9 moles 1.sup.-1 min.sup.-1."
DeLuca, Marlene and McElroy, W. D., Meth. Anal. Chem., 40/4:803-5 (1968) report that L-(+)-luciferin (LH) in an aqueous pH 9.0 solution is a highly fluorescent compound with an excitation level of 385 nm, and with emission at approximately 540 nm. In an aqueous solution the quantum yield is 0.62. LH is an unstable compound in basic aqueous solutions.
2-carbamyl-6-methoxybenzothiazole, an intermediate in the synthesis of LH, is reported in Methods of Enzymology, Vol. 57, p. 19. There was no fluorescence reported for this material and this was verified in our experiments.
B. Environmental Condition Measurement.
Because environmental factors are known to cause hydrolysis of phosphate groups, monitoring of the rate of hydrolysis may indirectly monitor various environmental conditions. For example, extremes in temperature or pH, or metals may act as hydrolyzing forces. Accordingly, it is of value to have a fluorescent compound which is inhibited by attachment of a chemical moiety and which, upon cleavage of the chemical moiety by hydrolyzing forces, exhibits restored fluorescence.
The use of colorimetric tests for the presence of oxygen is known in the art, e.g., colorimetric tests for anaerobic environments. Fluorescent compounds may also be used for the measurement of oxygen level. Generally, where fluorescent compounds possess characteristic "long lifetime", the compound is capable of being quenched by the presence of oxygen. This occurs as electrons in the fluorescent compound drop down to a lower energy level as they emit light energy. If the time period in which the electrons drop is sufficient, some of the energy given off by the falling electron is harnessed by oxygen molecules. The minimum "lifetime" for the falling electron is approximately 10.sup.-15 seconds, but a longer lifetime provides for a more sensitive oxygen measurement. Accordingly, it is of value to have a fluorescent compound with "long lifetime" in order to measure oxidation.
C. Direct Detection, Assaying or Monitoring of Biological Molecules.
Labels for biological ligands are well known in the art, and these include radioactive substances, colorimetric indicators and fluorescent compounds. Typically, these substances are either incorporated into the biological ligand, as in the use of radioactive nucleotides, or are chemically attached to the ligand, as in the use of glutaraldehyde or various chemical "extension arms" which are used to attach fluorescent labels to antibodies.
Accordingly, the present invention provides the following advantages:
1. Fluorescent compounds which maintain stability in an aqueous environment; PA1 2. Fluorescent compounds which are easily detectable above background interference; PA1 3. Fluorescent compounds, which, upon attachment of a chemical moiety, severely decrease in fluorescence but, upon removal of said chemical moiety, are strongly fluorescent; PA1 4. Fluorescent compounds which exhibit a Stokes' shift sufficient for use as an assay indication or other marker; Fluorescent compounds which provide means for detection of at least about 10 attomolar (10.sup.-18 molar) concentrations of alkaline phosphatase; PA1 6. Fluorescent compounds which possess "long lifetime" and provide means of detecting oxidizing agents; PA1 7. Fluorescent compounds which maintain fluorescence characteristics in a variety of solvents; PA1 8. Non-fluorescent phosphate compounds which are stable in water which can form fluorescent compounds upon hydrolysis; PA1 9. A class of fluorescent compounds of which some members can be excited with visible light. PA1 b) The carbon at position 2 is linked to a chemical moiety comprised of at least two atoms which extend resonance of the benzothiazole ring system; and, PA1 c) A nitrogen atom is located at position 3; and PA1 d) A sulfur atom is located at position 1. PA1 ABT: 2-carbamoyl-6-hydroxybenzothiazole PA1 BBT: 2'-(2-benzothiazolyl)-6'-hydroxybenzothiazole PA1 CBT: 2-cyano-6-hydroxybenzothiazole PA1 Stokes' shift: a physical constant that is characteristic of luminescent molecules which is the difference between the wavelength of the excitation and emission maxima. PA1 Rayleigh-Scatter: interference due to light emitted as a result of electron vibration due to excitation by photon energy. PA1 Raman: appears in fluorescence spectra at higher and lower wavelengths than the Rayleigh-scatter peak. These Raman bands are satellites of the Rayleigh-scatter peak with a constant frequency difference from the exciting radiation. These bands are due to vibrational energy being added to, or subtracted from, this excitation photon. PA1 Fluorescence efficiency: the amount of light emitted as a proportion of that used to excite. PA1 Excitation wavelength: the wavelength of light used to generate fluorescence emission, measured in arbitrary units. PA1 Emission wavelength: the wavelength of light emitted by a fluorescent molecule after excitation. PA1 Km: the substrate concentration at which the velocity of the enzymatic reaction is half maximal. PA1 Turnover number: the number of substrate molecules transformed per unit time by a single enzyme molecule when the enzyme is the rate limiting factor. PA1 Molar absorptivity: the intensity of an absorption band in the ultraviolet or visible spectrum. PA1 Substrate: the molecule on which the enzyme exerts a catalytic action. PA1 Enzyme: catalyst capable of greatly enhancing the rate of specific chemical reactions. PA1 Resonance: when the contribution of each of several structures is to be weighted in some way that accords with the degree of bonding each would have if it represented an actual molecule with the specified geometry.